Maternally transcribed protein

ABSTRACT

The invention provides a human maternally transcribed protein (HMTP) and polynucleotides which identify and encode HMTP. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. The invention also provides methods for treating disorders associated with expression of HMTP.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of a newhuman embryogenesis protein and to the use of these sequences in thediagnosis, prevention, and treatment of inflammation and disordersassociated with cell proliferation and apoptosis.

BACKGROUND OF THE INVENTION

Mammalian embryogenesis is a process which encompasses the first fewweeks of development following conception. During this period,embryogenesis proceeds from a single fertilized egg to the formation ofthe three embryonic tissues, then to an embryo which has most of itsinternal organs and all of its external features.

The normal course of mammalian embryogenesis depends on the correcttemporal and spatial regulation of a large number of genes and tissues.These regulation processes have been intensely studied in mouse. Forexample, spindlin (Spin) is an abundant maternal transcript present inthe unfertilized egg and 2-cell stage embryo. Spindlin functions incell-cycle regulation during the transition from gamete to embryo; itsactivity is modulated by cell cycle-dependent phosphorylation (Oh, B. etal. (1997) Development 124: 493-503). Another maternal transcript, Mem3is abundant in the unfertilized egg and the newly formed zygote (Hwang,S. et al. (1996) Mamm. Genome 7: 586-590). The amino acid sequence ofMem3 resembles the yeast vacuolar protein sorting 35 (VPS35), a proteininvolved in the intracellular sorting of proteases and hydrolases. Manyof the lysosomal storage diseases in human and other mammals appear tobe caused by incorrect sorting of the lysosomal hydrolases. Stillanother maternally transcribed protein, Maid, is shown to be a negativeregulator of basic helix-loop-helix (bHLH) proteins (GI 1255172). bHLHproteins are a group of transcription factors which play a pivotal rolein cell differentiation, tissue-specific determination, andembryogenesis. Dysregulated expression or loss of function of these bHLHprotein have been correlated with the development of leukemia and tumors(Gitelman, I. (1997) Dev. Biol. 189: 205-214; Rostomily, R. C. et al.(1997) Cancer Res. 57: 3526-3531).

The discovery of a new human maternally transcribed protein and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, prevention, andtreatment of inflammation and disorders associated with cellproliferation and apoptosis.

SUMMARY OF THE INVENTION

The invention features a substantially purified polypeptide, thematernally transcribed protein (HMTP), having the amino acid sequenceshown in SEQ ID NO:1, or fragments thereof.

The invention further provides an isolated and substantially purifiedpolynucleotide sequence encoding the polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or fragments thereof and a compositioncomprising said polynucleotide sequence. The invention also provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence encoding the amino acid sequence SEQ IDNO:1, or fragments of said polynucleotide sequence. The inventionfurther provides a polynucleotide sequence comprising the complement ofthe polynucleotide sequence encoding the amino acid sequence of SEQ IDNO:1, or fragments or variants of said polynucleotide sequence.

The invention also provides an isolated and purified sequence comprisingSEQ ID NO:2 or variants thereof. In addition, the invention provides apolynucleotide sequence which hybridizes under stringent conditions tothe polynucleotide sequence of SEQ ID NO:2. The invention also providesa polynucleotide sequence comprising the complement of SEQ ID NO:2 orfragments or variants thereof.

The present invention further provides an expression vector containingat least a fragment of any of the claimed polynucleotide sequences. Inyet another aspect, the expression vector containing the polynucleotidesequence is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment thereof,the method comprising the steps of: a) culturing the host cellcontaining an expression vector containing at least a fragment of thepolynucleotide sequence encoding HMTP under conditions suitable for theexpression of the polypeptide; and b) recovering the polypeptide fromthe host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified HMTP having the amino acid sequence of SEQ IDNO:1 in conjunction with a suitable pharmaceutical carrier.

The invention also provides a purified antagonist of the polypeptide ofSEQ ID NO:1. In one aspect the invention provides a purified antibodywhich binds to a polypeptide comprising the amino acid sequence of SEQID NO:1.

Still further, the invention provides a purified agonist of thepolypeptide of SEQ ID NO:1.

The invention also provides a method for stimulating cell proliferationcomprising administering to a cell an effective amount of purified HMTP.

The invention also provides a method for preventing or treating adisorder associated with an increase in apoptosis comprisingadministering to a subject in need of such treatment an effective amountof a pharmaceutical composition comprising purified HMTP.

The invention also provides a method for preventing or treating a cancercomprising administering to a subject in need of such treatment aneffective amount of an antagonist of HMTP.

The invention also provides a method for preventing or treating aninflammation comprising administering to a subject in need of suchtreatment an effective amount of an antagonist of HMTP.

The invention also provides a method for detecting a polynucleotidewhich encodes HMTP in a biological sample comprising the steps of: a)hybridizing the complement of the polynucleotide sequence which encodesSEQ ID NO:1 to nucleic acid material of a biological sample, therebyforming a hybridization complex; and b) detecting the hybridizationcomplex, wherein the presence of the complex correlates with thepresence of a polynucleotide encoding HMTP in the biological sample. Inone aspect the nucleic acid material of the biological sample isamplified by the polymerase chain reaction prior to hybridization.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, 1D, and 1E show the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:2) of HMTP. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering Co.Ltd. San Bruno, Calif.).

FIG. 2 shows the amino acid sequence alignments between HMTP (2075107;SEQ ID NO:1) and a mouse maternally transcribed protein, Maid (GI1255172; SEQ ID NO:3), produced using the multisequence alignmentprogram of DNASTAR software (DNASTAR Inc., Madison Wis.).

FIGS. 3A and 3B show the hydrophobicity plots for HMTP (SEQ ID NO:1) andthe mouse Maid (SEQ ID NO:3), respectively. The positive X axis reflectsamino acid position, and the negative Y axis reflects hydrophobicity(MACDNASIS PRO software).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "an", and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to "ahost cell" includes a plurality of such host cells, reference to the"antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

Definitions

HMTP, as used herein, refers to the amino acid sequences ofsubstantially purified HMTP obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

The term "agonist", as used herein, refers to a molecule which, whenbound to HMTP, increases or prolongs the duration of the effect of HMTP.Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to and modulate the effect of HMTP.

An "allele" or "allelic sequence", as used herein, is an alternativeform of the gene encoding HMTP. Alleles may result from at least onemutation in the nucleic acid sequence and may result in altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

"Altered" nucleic acid sequences encoding HMTP as used herein includethose with deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent HMTP. Included within this definition arepolymorphisms which may or may not be readily detectable using aparticular oligonucleotide probe of the polynucleotide encoding HMTP,and improper or unexpected hybridization to alleles, with a locus otherthan the normal chromosomal locus for the polynucleotide sequenceencoding HMTP. The encoded protein may also be "altered" and containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent HMTP.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological or immunological activity of HMTP is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline, glycine and alanine, asparagine and glutamine, serine andthreonine, and phenylalanine and tyrosine.

"Amino acid sequence" as used herein refers to an oligopeptide, peptide,polypeptide, or protein sequence, and fragment thereof, and to naturallyoccurring or synthetic molecules. Fragments of HMTP are preferably about5 to about 15 amino acids in length and retain the biological activityor the immunological activity of HMTP. Where "amino acid sequence" isrecited herein to refer to an amino acid sequence of a naturallyoccurring protein molecule, amino acid sequence, and like terms, are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

"Amplification" as used herein refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term "antagonist" as used herein, refers to a molecule which, whenbound to HMTP, decreases the amount or the duration of the effect of thebiological or immunological activity of HMTP. Antagonists may includeproteins, nucleic acids, carbohydrates, antibodies or any othermolecules which decrease the effect of HMTP.

As used herein, the term "antibody" refers to intact molecules as wellas fragments thereof, such as Fab, F(ab')₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind HMTP polypeptidescan be prepared using intact polypeptides or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide oroligopeptide used to immunize an animal can be derived from thetranslation of RNA or synthesized chemically and can be conjugated to acarrier protein, if desired. Commonly used carriers that are chemicallycoupled to peptides include bovine serum albumin and thyroglobulin,keyhole limpet hemocyanin. The coupled peptide is then used to immunizethe animal (e.g., a mouse, a rat, or a rabbit).

The term "antigenic determinant", as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The term "antisense", as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term "antisense strand" is used in reference toa nucleic acid strand that is complementary to the "sense" strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation "negative" is sometimes used in referenceto the antisense strand, and "positive" is sometimes used in referenceto the sense strand.

The term "biologically active", as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic HMTP, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms "complementary" or "complementarity", as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence"A-G-T" binds to the complementary sequence "T-C-A". Complementaritybetween two single-stranded molecules may be "partial", in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands and in thedesign and use of PNA molecules.

A "composition comprising a given polynucleotide sequence" as usedherein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise a dry formulationor an aqueous solution. Compositions comprising polynucleotide sequencesencoding HMTP (SEQ ID NO:1) or fragments thereof (e.g., SEQ ID NO:2 andfragments thereof) may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS) and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

"Consensus", as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, has been extended usingXL-PCR (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3' directionand resequenced, or has been assembled from the overlapping sequences ofmore than one Incyte Clone using a computer program for fragmentassembly (e.g., GELVIEW fragment assembly system, GCG, Madison, Wis.).Some sequences have been both extended and assembled to produce theconsensus sequence.

The term "correlates with expression of a polynucleotide", as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO:2 by northern analysis is indicative of thepresence of mRNA encoding HMTP in a sample and thereby correlates withexpression of the transcript from the polynucleotide encoding theprotein.

A "deletion", as used herein, refers to a change in the amino acid ornucleotide sequence and results in the absence of one or more amino acidresidues or nucleotides.

The term "derivative", as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to HMTP or theencoded HMTP. Such modifications include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

The term "homology", as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term "substantiallyhomologous." The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

Human artificial chromosomes (HACs) are linear microchromosomes whichmay contain DNA sequences of 10K to 10M in size and contain all of theelements required for stable mitotic chromosome segregation andmaintenance (Harrington, J. J. et al. (1997) Nat. Genet. 15:345-355).

The term "humanized antibody", as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

The term "hybridization", as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term "hybridization complex", as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

An "insertion" or "addition", as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

"Microarray" refers to an array of distinct polynucleotides oroligonucleotides synthesized on a substrate, such as paper, nylon orother type of membrane, filter, chip, glass slide, or any other suitablesolid support.

The term "modulate", as used herein, refers to a change in the activityof HMTP. For example, modulation may cause an increase or a decrease inprotein activity, binding characteristics, or any other biological,functional or immunological properties of HMTP.

"Nucleic acid sequence" as used herein refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNAof genomic or synthetic origin which may be single- or double-stranded,and represent the sense or antisense strand. "Fragments" are thosenucleic acid sequences which are greater than 60 nucleotides than inlength, and most preferably includes fragments that are at least 100nucleotides or at least 1000 nucleotides, and at least 10,000nucleotides in length.

The term "oligonucleotide" refers to a nucleic acid sequence of at leastabout 6 nucleotides to about 60 nucleotides, preferably about 15 to 30nucleotides, and more preferably about 20 to 25 nucleotides, which canbe used in PCR amplification or a hybridization assay, or a microarray.As used herein, oligonucleotide is substantially equivalent to the terms"amplimers","primers", "oligomers", and "probes", as commonly defined inthe art.

"Peptide nucleic acid", PNA as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast five nucleotides in length linked to a peptide backbone of aminoacid residues which ends in lysine. The terminal lysine conferssolubility to the composition. PNAs may be pegylated to extend theirlifespan in the cell where they preferentially bind complementary singlestranded DNA and RNA and stop transcript elongation (Nielsen, P. E. etal. (1993) Anticancer Drug Des. 8:53-63).

The term "portion", as used herein, with regard to a protein (as in "aportion of a given protein") refers to fragments of that protein. Thefragments may range in size from five amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein "comprising atleast a portion of the amino acid sequence of SEQ ID NO:1" encompassesthe full-length HMTP and fragments thereof.

The term "sample", as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding HMTP, orfragments thereof, or HMTP itself may comprise a bodily fluid, extractfrom a cell, chromosome, organelle, or membrane isolated from a cell, acell, genomic DNA, RNA, or cDNA (in solution or bound to a solidsupport, a tissue, a tissue print, and the like).

The terms "specific binding" or "specifically binding", as used herein,refers to that interaction between a protein or peptide and an agonist,an antibody and an antagonist. The interaction is dependent upon thepresence of a particular structure (i.e., the antigenic determinant orepitope) of the protein recognized by the binding molecule. For example,if an antibody is specific for epitope "A", the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled "A" and the antibody will reduce the amount of labeled A boundto the antibody.

The terms "stringent conditions" or "stringency", as used herein, referto the conditions for hybridization as defined by the nucleic acid,salt, and temperature. These conditions are well known in the art andmay be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors be may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

The term "substantially purified", as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

A "substitution", as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

"Transformation", as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such"transformed" cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

A "variant" of HMTP, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,e.g., replacement of a glycine with a tryptophan. Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

The Invention

The invention is based on the discovery of a new human maternallytranscribed protein (hereinafter referred to as "HMTP"), thepolynucleotides encoding HMTP, and the use of these compositions for thediagnosis, prevention, or treatment of inflammation and disordersassociated with cell proliferation and apoptosis.

Nucleic acids encoding the HMTP of the present invention were firstidentified in Incyte Clone 2075107 from a pancreatic islet cell cDNAlibrary (ISLTNOT01) using a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:2, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 2075107 (ISLTNOT01), 2220980 (LUNGNOT18), 2082077 (UTRSNOT08),1988474 (LUNGAST01), 1988326 (LUNGAST01), 1902619 (OVARNOT07), 1853232(LUNGFET03), 1691201 (PROSTUT10), 1641859 (HEARFET01).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, 1C,1D, and 1E. HMTP is 310 amino acids in length. HMTP has one potentialamidation site encompassing residues N215-K218; two potentialN-glycosylation sites encompassing residues N156-E159 and N161-D164; sixpotential casein kinase II phosphorylation sites encompassing residuesS31-E34, S177-D180, S184-D187, S237-D240, T304-E307, and S306-E309; andfour potential protein kinase C phosphorylation sites encompassingresidues T35-K37, T63-R65, T253-R255, and S258-K260. As shown in Figure2, HMTP has chemical and structural homology with a mouse maternallytranscribed protein, Maid (GI 1255172; SEQ ID NO:3). In particular, HMTPshares 62% identity with Maid. HMTP and Maid also exhibit similarhydrophobicity, as shown in Figures 3A and 3B. Northern analysis showsthe expression of HMTP in various cDNA libraries, at least 40% of whichare immortalized or cancerous, at least 28% of which involve immuneresponse, and at least 25% of which involve fetal/infant development.

The invention also encompasses HMTP variants. A preferred HMTP variantis one having at least 80%, and more preferably at least 90%, amino acidsequence identity to the HMTP amino acid sequence (SEQ ID NO:1) andwhich retains at least one biological, immunological or other functionalcharacteristic or activity of HMTP. A most preferred HMTP variant is onehaving at least 95% amino acid sequence identity to SEQ ID NO:1.

The invention also encompasses polynucleotides which encode HMTP.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of HMTP can be used to produce recombinant molecules whichexpress HMTP. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A, 1B, 1C, 1D, and 1E.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding HMTP, some bearing minimal homology to the nucleotide sequencesof any known and naturally occurring gene, may be produced. Thus, theinvention contemplates each and every possible variation of nucleotidesequence that could be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet genetic code as applied to the nucleotide sequence ofnaturally occurring HMTP, and all such variations are to be consideredas being specifically disclosed.

Although nucleotide sequences which encode HMTP and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HMTP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HMTP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding HMTP and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or fragmentsthereof, which encode HMTP and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding HMTP or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, under various conditions ofstringency as taught in Wahl, G. M. and S. L. Berger (1987; MethodsEnzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol.152:507-511).

Methods for DNA sequencing which are well known and generally availablein the art and may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE (U.S. Biochemical Corp, Cleveland, Ohio),Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE amplification system(Gibco/BRL, Gaithersburg, Md.). Preferably, the process is automatedwith machines such as the MICROLAB 2200 (Hamilton, Reno, Nev.), Peltierthermal cycler (PTC200; M.J. Research, Watertown, Mass.), and the ABICATALYST and 373 and 377 DNA sequencers (Perkin Elmer).

The nucleic acid sequences encoding HMTP may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,"restriction-site" PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed usingcommercially available software such as OLIGO 4.06 primer analysissoftware (National Biosciences Inc., Plymouth, Minn.), or anotherappropriate program, to be 22-30 nucleotides in length, to have a GCcontent of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. The method uses several restrictionenzymes to generate a suitable fragment in the known region of a gene.The fragment is then circularized by intramolecular ligation and used asa PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5' non-transcribedregulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devisecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. GENOTYPER and SEQUENCE NAVIGATOR,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode HMTP may be used in recombinant DNAmolecules to direct expression of HMTP, fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressHMTP.

As will be understood by those of skill in the art, it may beadvantageous to produce HMTP-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter HMTP encodingsequences for a variety of reasons, including but not limited to,alterations which modify the cloning, processing, and/or expression ofthe gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HMTP may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of HMTP activity, it may be useful toencode a chimeric HMTP protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the HMTP encoding sequence and theheterologous protein sequence, so that HMTP may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding HMTP may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223;Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of HMTP, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A peptide synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W. H. Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of HMTP, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active HMTP, the nucleotide sequencesencoding HMTP or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding HMTP andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding HMTP. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector--enhancers, promoters, 5' and 3'untranslated regions--which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding HMTP,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for HMTP. For example, when largequantities of HMTP are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding HMTP may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding HMTP may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196.

An insect system may also be used to express HMTP. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding HMTP may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of HMTP will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which HMTP may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding HMTP may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing HMTP in infected host cells (Logan, J. and Shenk,T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of 6 to 10M are constructed and delivered via conventionaldelivery methods (liposomes, polycationic amino polymers, or vesicles)for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding HMTP. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding HMTP, its initiation codon, and upstream sequences are insertedinto the appropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including the ATG initiation codon shouldbe provided. Furthermore, the initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons may be of various origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used, such as those described in the literature(Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and W138), are available from the American TypeCulture Collection (ATCC; Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressHMTP may be transformed using expression vectors which may contain viralorigins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr, which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14); and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, βglucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding HMTP isinserted within a marker gene sequence, transformed cells containingsequences encoding HMTP can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding HMTP under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding HMTP and express HMTP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

The presence of polynucleotide sequences encoding HMTP can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding HMTP. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding HMTP to detect transformantscontaining DNA or RNA encoding HMTP.

A variety of protocols for detecting and measuring the expression ofHMTP, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson HMTP is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding HMTP includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding HMTP, or anyfragments thereof may be cloned into a vector for the production of anmRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn (Kalamazoo, Mich.);Promega (Madison, Wis.); and U.S. Biochemical Corp. Cleveland, Ohio)).Suitable reporter molecules or labels, which may be used for ease ofdetection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding HMTP may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeHMTP may be designed to contain signal sequences which direct secretionof HMTP through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding HMTP to nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and HMTP may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingHMTP and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMIAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying HMTP from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

In addition to recombinant production, fragments of HMTP may be producedby direct peptide synthesis using solid-phase techniques (Merrifield, J.(1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may beperformed using manual techniques or by automation. Automated synthesismay be achieved, for example, using an Applied Biosystems 431A peptidesynthesizer (Perkin Elmer). Various fragments of HMTP may be chemicallysynthesized separately and combined using chemical methods to producethe full length molecule.

Therapeutics

Chemical and structural homology exists between HMTP and a mousematernally transcribed protein, Maid (GI 1255172; SEQ ID NO:3). Northernanalysis shows that the expression of HMTP is associated with cancer,inflammation, and fetal/infant development.

During fetal development, decreased expression of HMTP may cause anincrease in apoptosis with no adverse effects to the subject. However,in other situations and in adults, decreased expression of HMTP maycause an increase in apoptosis which may have detrimental effects.Therefore, in one embodiment, HMTP or a fragment or derivative thereofmay be administered to a subject to prevent or treat a disorderassociated with an increase in apoptosis. Such disorders include, butare not limited to, AIDS and other infectious or geneticimmunodeficiencies, neurodegenerative diseases such as Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa, and cerebellar degeneration, myelodysplastic syndromes suchas aplastic anemia, ischemic injuries such as myocardial infarction,stroke, and reperfusion injury, toxin-induced diseases such asalcohol-induced liver damage, cirrhosis, and lathyrism, wasting diseasessuch as cachexia, viral infections such as those caused by hepatitis Band C, and osteoporosis.

In another embodiment, a pharmaceutical composition comprising purifiedHMTP may be used to prevent or treat a disorder associated withincreased apoptosis including, but not limited to, those listed above.

In still another embodiment, an agonist which is specific for HMTP maybe used to prevent or treat a disorder associated with increasedapoptosis including, but not limited to, those listed above.

In still another embodiment, a vector capable of expressing HMTP, or afragment or a derivative thereof, may be used to prevent or treat adisorder associated with increased apoptosis including, but not limitedto, those listed above.

In a further embodiment, HMTP or a fragment or derivative thereof may beadded to cells to stimulate cell proliferation. In particular, HMTP maybe added to a cell or cells in vivo using delivery mechanisms such asliposomes, viral based vectors, or electroinjection for the purpose ofpromoting regeneration or cell differentiation of the cell or cells. Inaddition, HMTP may be added to a cell, cell line, tissue or organculture in vitro or ex vivo to stimulate cell proliferation for use inheterologous or autologous transplantation. In some cases, the cell willhave been selected for its ability to fight an infection or a cancer orto correct a genetic defect in a disease such as sickle cell anemia, Pthalassemia, cystic fibrosis, or Huntington's chorea.

In another further embodiment, an agonist which is specific for HMTP maybe administered to a cell to stimulate cell proliferation, as describedabove.

In another further embodiment, a vector capable of expressing HMTP, or afragment or a derivative thereof, may be administered to a cell or cellsin vivo using delivery mechanisms, or to a cell to stimulate cellproliferation, as described above.

Increased expression of HMTP appears to be associated with increasedcell proliferation. Therefore, in one embodiment, an antagonist of HMTP,or a fragment or a derivative thereof, may be administered to a subjectto prevent or treat cancer. Such disorders include various types ofcancer including, but not limited to, adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, andparticularly, cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus. In one aspect, an antibody specific for HMTP may be useddirectly as an antagonist, or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress HMTP.

In still another embodiment, a vector expressing the complement of thepolynucleotide encoding HMTP, or a fragment or a derivative thereof, maybe administered to a subject to prevent or treat a disorder associatedwith cell proliferation including, but not limited to, the types ofcancer listed above.

In another embodiment, an antagonist of HMTP, or a fragment or aderivative thereof, may be administered to a subject to prevent or treatinflammation. Such inflammation includes, but is not limited to, thatwhich is associated with disorders such as Addison's disease, adultrespiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, atrophic gastritis, glomerulonephritis, gout, Graves'disease, hypereosinophilia, irritable bowel syndrome, lupuserythematosus, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections; and trauma. In one aspect, anantibody specific for HMTP may be used directly as an antagonist, orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express HMTP.

In still another embodiment, a vector expressing the complement of thepolynucleotide encoding HMTP, or a fragment or a derivative thereof, maybe administered to a subject to prevent or treat inflammation associatedwith any disorder including, but not limited to, those listed above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of HMTP may be produced using methods which are generallyknown in the art. In particular, purified HMTP may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind HMTP.

Antibodies to HMTP may be generated using methods that are well known inthe art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith HMTP or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to HMTP have an amino acid sequence consisting of atleast five amino acids and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of HMTP amino acids may be fused with those of another proteinsuch as keyhole limpet hemocyanin and antibody produced against thechimeric molecule.

Monoclonal antibodies to HMTP may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.62:109-120).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceHMTP-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter,G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for HMTP mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between HMTP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering HMTP epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingHMTP, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, the complement of the polynucleotide encodingHMTP may be used in situations in which it would be desirable to blockthe transcription of the mRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding HMTP. Thus,complementary molecules or fragments may be used to modulate HMTPactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligonucleotides orlarger fragments, can be designed from various locations along thecoding or control regions of sequences encoding HMTP.

Expression vectors derived from retro viruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencewhich is complementary to the polynucleotides of the gene encoding HMTP.These techniques are described both in Sambrook et al. (supra) and inAusubel et al. (supra).

Genes encoding HMTP can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes HMTP. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5' or regulatory regions of the gene encoding HMTP(signal sequence, promoters, enhancers, and introns). Oligonucleotidesderived from the transcription initiation site, e.g., between positions-10 and +10 from the start site, are preferred. Similarly, inhibitioncan be achieved using "triple helix" base-pairing methodology. Triplehelix pairing is useful because it causes inhibition of the ability ofthe double helix to open sufficiently for the binding of polymerases,transcription factors, or regulatory molecules. Recent therapeuticadvances using triplex DNA have been described in the literature (Gee,J. E. et al. (1994) In: Huber, B. E. and B. I. Carr, Molecular andImmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). Thecomplementary sequence or antisense molecule may also be designed toblock translation of mRNA by preventing the transcript from binding toribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding HMTP.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding HMTP. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA constitutivelyor inducibly can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2'O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections orpolycationic amino polymers (Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-66; incorporated herein by reference) may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of HMTP, antibodies toHMTP, mimetics, agonists, antagonists, or inhibitors of HMTP. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Non-lipid polycationicamino polymers may also be used for delivery. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acids, etc.Salts tend to be more soluble in aqueous or other protonic solvents thanare the corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, ata pH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of HMTP, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example HMTP or fragments thereof, antibodies of HMTP,agonists, antagonists or inhibitors of HMTP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, which can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind HMTP may beused for the diagnosis of conditions or diseases characterized byexpression of HMTP, or in assays to monitor patients being treated withHMTP, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for HMTP includemethods which utilize the antibody and a label to detect HMTP in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuring HMTPare known in the art and provide a basis for diagnosing altered orabnormal levels of HMTP expression. Normal or standard values for HMTPexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toHMTP under conditions suitable for complex formation. The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric, means. Quantities of HMTP expressed insubject, samples from biopsied tissues are compared with the standardvalues. Deviation between standard and subject values establishes theparameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingHMTP may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofHMTP may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of HMTP,and to monitor regulation of HMTP levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HMTP or closely related molecules, may be used to identifynucleic acid sequences which encode HMTP. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5' regulatory region, or a less specific region,e.g., especially in the 3' coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding HMTP, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe HMTP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring HMTP.

Means for producing specific hybridization probes for DNAs encoding HMTPinclude the cloning of nucleic acid sequences encoding HMTP or HMTPderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding HMTP may be used for the diagnosis ofconditions or disorders which are associated with expression of HMTP.Examples of such conditions or disorders include, but are not limitedto, disorders associated with cell proliferation such as adenocarcinoma,leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, andparticularly, cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus; disorders associated with inflammation such as Addison'sdisease, adult respiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitus, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, atrophic gastritis, glomerulonephritis, gout, Graves'disease, hypereosinophilia, irritable bowel syndrome, lupuserythematosus, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation,osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis,extracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections; and trauma; disorders associatedwith apoptosis such as AIDS and other infectious or geneticimmunodeficiencies, neurodegenerative diseases such as Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, retinitispigmentosa, and cerebellar degeneration, myelodysplastic syndromes suchas aplastic anemia, ischemic injuries such as myocardial infarction,stroke, and reperfusion injury, toxin-induced diseases such asalcohol-induced liver damage, cirrhosis, and lathyrism, wasting diseasessuch as cachexia, viral infections such as those caused by hepatitis Band C, and osteoporosis. The polynucleotide sequences encoding HMTP maybe used in Southern or northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; or in dipstick, pin,ELISA assays or microarrays utilizing fluids or tissues from patientbiopsies to detect altered HMTP expression. Such qualitative orquantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding HMTP may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingHMTP may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding HMTP in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of HMTP, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes HMTP, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding HMTP may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably consist of two nucleotide sequences,one with sense orientation (5'->3') and another with antisense (3'-<5'),employed under optimized conditions for identification of a specificgene or condition. The same two oligomers, nested sets of oligomers, oreven a degenerate pool of oligomers may be employed under less stringentconditions for detection and/or quantitation of closely related DNA orRNA sequences.

Methods which may also be used to quantitate the expression of HMTPinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.212:229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

In further embodiments, an oligonucleotide derived from any of thepolynucleotide sequences described herein may be used as a target in amicroarray. The microarray can be used to monitor the expression levelof large numbers of genes simultaneously (to produce a transcriptimage), and to identify genetic variants, mutations and polymorphisms.This information will be useful in determining gene function,understanding the genetic basis of disease, diagnosing disease, and indeveloping and monitoring the activity of therapeutic agents (Heller, R.et al. (1997) Proc. Natl. Acad. Sci. 94:2150-55).

In one embodiment, the microarray is prepared and used according to themethods described in PCT application WO95/11995 (Chee et al.), Lockhart,D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena, M. et al.(1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of which areincorporated herein in their entirety by reference.

The microarray is preferably composed of a large number of unique,single-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6-60 nucleotides inlength, more preferably 15-30 nucleotides in length, and most preferablyabout 20-25 nucleotides in length. For a certain type of microarray, itmay be preferable to use oligonucleotides which are only 7-10nucleotides in length. The microarray may contain oligonucleotides whichcover the known 5', or 3', sequence, sequential oligonucleotides whichcover the full length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray may be oligonucleotides that are specific to a gene orgenes of interest in which at least a fragment of to the sequence isknown or that are specific to one or more unidentified cDNAs which arecommon to a particular cell type, developmental or disease state.

In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5' or more preferably at the 3' end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In certain situations it may beappropriate to use pairs of oligonucleotides on a microarray. The"pairs" will be identical, except for one nucleotide which preferably islocated in the center of the sequence. The second oligonucleotide in thepair (mismatched by one) serves as a control. The number ofoligonucleotide pairs may range from two to one million. The oligomersare synthesized at designated areas on a substrate using alight-directed chemical process. The substrate may be paper, nylon orother type of membrane, filter, chip, glass slide or any other suitablesolid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/25 1116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a "gridded" array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray, the RNA or DNAfrom a biological sample is made into hybridization probes. The mRNA isisolated, and cDNA is produced and used as a template to make antisenseRNA (aRNA). The aRNA is amplified in the presence of fluorescentnucleotides, and labeled probes are incubated with the microarray sothat the probe sequences hybridize to complementary oligonucleotides ofthe microarray. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray. The biological samples may be obtained from any bodilyfluids (such as blood, urine, saliva, phlegm, gastric juices, etc.),cultured cells, biopsies, or other tissue preparations. A detectionsystem may be used to measure the absence, presence, and amount ofhybridization for all of the distinct sequences simultaneously. Thisdata may be used for large scale correlation studies on the sequences,mutations, variants, or polymorphisms among samples.

In another embodiment of the invention, the nucleic acid sequences whichencode HMTP may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome, to a specific regionof a chromosome or to artificial chromosome constructions, such as humanartificial chromosomes (HACs), yeast artificial chromosomes (YACs),bacterial artificial chromosomes (BACs), bacterial P1 constructions orsingle chromosome cDNA libraries as reviewed in Price, C. M. (1993)Blood Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.

Fluorescent in situ hybridization (FISH as described in Verma et al.(1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York, N.Y.) may be correlated with other physical chromosome mappingtechniques and genetic map data. Examples of genetic map data can befound in various scientific journals or at Online Mendelian Inheritancein Man (OMIM). Correlation between the location of the gene encodingHMTP on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, HMTP, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenHMTP and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to HMTP large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with HMTP, or fragments thereof, and washed. Bound HMTP is thendetected by methods well known in the art. Purified HMTP can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding HMTP specificallycompete with a test compound for binding HMTP. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with HMTP.

In additional embodiments, the nucleotide sequences which encode HMTPmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I ISLTNOT01 cDNA Library Construction

The ISLTNOT01 cDNA library was constructed from total RNA isolated frommicroscopically normal pancreatic islet cells. The frozen tissues werehomogenized and lysed using a Polytron PT-3000 homogenizer (BrinkmannInstruments, Westbury, N.Y.) in guanidinium isothiocyanate solution. Thelysates were centrifuged over a 5.7 M CsCl cushion using a Beckman SW28rotor in a L8-70M ultracentrifuge (Beckman Instruments) for 18 hours at25,000 rpm at ambient temperature. The RNA was extracted with acidphenol pH 4.7, precipitated using 0.3 M sodium acetate and 2.5 volumesof ethanol, resuspended in RNase-free water, and DNase treated at 37° C.RNA extraction and precipitation were repeated as before. The mRNA wasthen isolated using the OLIGOTEX kit (QIAGEN, Chatsworth, Calif.) andused to construct the cDNA library.

The mRNA were handled according to the recommended protocols in theSUPERSCRIPT plasmid system for cDNA synthesis and plasmid cloning (Cat.#18248-013, Gibco/BRL). cDNAs were fractionated on a SEPHAROSE CL4Bcolumn (Cat. #275105-01, Pharmacia), and those cDNAs exceeding 400 bpwere ligated into pINCY 1 (Incyte Pharmaceuticals, Inc., Palo Alto, CA).The plasmid pINCY 1 was subsequently transformed into DH5α™ competentcells (Cat. #18258-012, Gibco/BRL).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the REAL PREP96 plasmid kit (Catalog #26173, QIAGEN). This kit enabled thesimultaneous purification of 96 samples in a 96-well block usingmulti-channel reagent dispensers. The recommended protocol was employedexcept for the following changes: 1) the bacteria were cultured in 1 mlof sterile Terrific Broth (Catalog #22711, GIBCO/BRL) with carbenicillinat 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours and at the end of incubation, the cells werelysed with 0.3 ml of lysis buffer; and 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol.Biol. 94:441f), using a MICROLAB 2200 (Hamilton, Reno, Nev.) incombination with Peltier thermal cyclers (PTC200 from M.J. Research,Watertown, Mass.) and Applied Biosystems 377 DNA sequencing systems; andthe reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences of the Sequence Listing or amino acid sequencesdeduced from them were used as query sequences against databases such asGenBank, SwissProt, BLOCKS, and Pima II. These databases which containpreviously identified and annotated sequences were searched for regionsof homology (similarity) using BLAST, which stands for Basic LocalAlignment Search Tool (Altschul SF (1993) J. Mol. Evol. 36:290-300;Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-10).

BLAST produces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal or plant) origin. Other algorithms such asthe one described in Smith R. F. and T. F. Smith (1992 ProteinEngineering 5:35-51), incorporated herein by reference, can be used whendealing with primary sequence patterns and secondary structure gappenalties. As disclosed in this application, the sequences have lengthsof at least 49 nucleotides, and no more than 12% uncalled bases (where Nis recorded rather than A, C, G, or T).

The BLAST approach, as detailed in Karlin and Altschul (1993; Proc NatAcad Sci 90:5873-7) and incorporated herein by reference, searchesmatches between a query sequence and a database sequence, to evaluatethe statistical significance of any matches found, and to report onlythose matches which satisfy the user-selected threshold of significance.In this application, threshold was set at 10⁻²⁵ for nucleotides and10⁻¹⁴ for peptides.

V Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. (1993)J.Mol.Evol. 36:290-300; Altschul, S. F. et al. (1990) J.Mol.Evol.215:403-410) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding HMTP occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of HMTP Encoding Polynucleotides

The nucleic acid sequence of the Incyte Clone 2075107 was used to designoligonucleotide primers for extending a partial nucleotide sequence tofull length. One primer was synthesized to initiate extension in theantisense direction, and the other was synthesized to extend sequence inthe sense direction. Primers were used to facilitate the extension ofthe known sequence "outward" generating amplicons containing new,unknown nucleotide sequence for the region of interest. The initialprimers were designed from the cDNA using OLIGO 4.06 software (NationalBiosciences), or another appropriate program, to be about 22 to about 30nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures of about 68° to about 72°C. Any stretch of nucleotides which would result in hairpin structuresand primer-primer dimerizations was avoided.

Selected human cDNA libraries (Gibco/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. Beginning with 40 pmol of each primer and the recommendedconcentrations of all other components of the kit, PCR was performedusing the Peltier thermal cycler (PTC200; M. J. Research, Watertown,Mass.) and the following parameters:

    ______________________________________                                        Step 1       94° C. for 1 min (initial denaturation)                   Step 2       65° C. for 1 min                                          Step 3       68° C. for 6 min                                          Step 4       94° C. for 15 sec                                         Step 5       65° C. for 1 min                                          Step 6       68° C. for 7 min                                          Step 7       Repeat step 4-6 for 15 additional cycles                         Step 8       94° C. for 15 sec                                         Step 9       65° C. for 1 min                                          Step 10      68° C. for 7:15 min                                       Step 11      Repeat step 8-10 for 12 cycles                                   Step 12      72° C. for 8 min                                          Step 13      4° C. (and holding)                                       ______________________________________                                    

A 5-10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products were excised from the gel,purified using QIAQUICK (QIAGEN Inc., Chatsworth, Calif.), and trimmedof overhangs using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) were transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB)-agar (Sambrook et al., supra) containing 2× Carb.The following day, several colonies were randomly picked from each plateand cultured in 150 μl of liquid LB/2× Carb medium placed in anindividual well of an appropriate, commercially-available, sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and afterdilution 1:10 with water, 5 μl of each sample was transferred into a PCRarray.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

    ______________________________________                                        Step 1     94° C. for 60 sec                                           Step 2     94° C. for 20 sec                                           Step 3     55° C. for 30 sec                                           Step 4     72° C. for 90 sec                                           Step 5     Repeat steps 2-4 for an additional 29 cycles                       Step 6     72° C. for 180 sec                                          Step 7     4° C. (and holding)                                         ______________________________________                                    

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:2 is used to obtain5' regulatory sequences using the procedure above, oligonucleotidesdesigned for 5' extension, and an appropriate genomic library.

VI Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences), labeled by combining50 pmol of each oligomer and 250 μCi of [γ-³² P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN, Boston, Mass.). Thelabeled oligonucleotides are substantially purified with SEPHADEX G-25superfine resin column (Pharmacia & Upjohn). A aliquot containing 10⁷counts per minute of the labeled probe is used in a typicalmembrane-based hybridization analysis of human genomic DNA digested withone of the following endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1,or Pvu II; DuPont NEN).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (NYTRAN PLUS, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots or the blots areexposed in a Phosphoimager cassette (Molecular Dynamics, Sunnyvale,Calif.), hybridization patterns are compared visually.

VII Microarrays

To produce oligonucleotides for a microarray, the nucleotide sequencedescribed herein is examined using a computer algorithm which starts atthe 3' end of the nucleotide sequence. The algorithm identifiesoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that would interfere with hybridization. Thealgorithm identifies 20 sequence-specific oligonucleotides of 20nucleotides in length (20-mers). A matched set of oligonucleotides iscreated in which one nucleotide in the center of each sequence isaltered. This process is repeated for each gene in the microarray, anddouble sets of twenty 20 mers are synthesized and arranged on thesurface of the silicon chip using a light-directed chemical process(Chee, M. et al., PCT/WO95/11995, incorporated herein by reference).

In the alternative, a chemical coupling procedure and an ink jet deviceare used to synthesize oligomers on the surface of a substrate(Baldeschweiler, J. D. et al., PCT/WO95/25116, incorporated herein byreference). In another alternative, a "gridded" array analogous to a dot(or slot) blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array may beproduced by hand or using available materials and machines and containgrids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots.After hybridization, the microarray is washed to remove nonhybridizedprobes, and a scanner is used to determine the levels and patterns offluorescence. The scanned images are examined to determine degree ofcomplementarity and the relative abundance of each oligonucleotidesequence on the microarray.

VIII Complementary Polynucleotides

Sequence complementary to the HMTP-encoding sequence, or any partthereof, is used to decrease or inhibit expression of naturallyoccurring HMTP. Although use of oligonucleotides comprising from about15 to about 30 base-pairs is described, essentially the same procedureis used with smaller or larger sequence fragments. Appropriateoligonucleotides are designed using OLIGO 4.06 software and the codingsequence of HMTP, SEQ ID NO:1. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5' sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the HMTP-encoding transcript.

IX Expression of HMTP

Expression of HMTP is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express HMTP in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion ofHMTP into the bacterial growth media which can be used directly in thefollowing assay for activity.

X Demonstration of HMTP Activity

HMTP can be expressed by transforming a mammalian cell line such asCOS7, HeLa or CHO with an eukaryotic expression vector encoding HMTP.Eukaryotic expression vectors are commercially available, and thetechniques to introduce them into cells are well known to those skilledin the art. The cells are incubated for 48-72 hours after transformationunder conditions appropriate for the cell line to allow expression ofHMTP. Then, phase microscopy is used to compare the mitotic index oftransformed versus control cells. An increase in the mitotic indexindicates HMTP activity.

XI Production of HMTP Specific Antibodies

HMTP that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc.) to determine regions of high immunogenicity anda corresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Selection of appropriateepitopes, such as those near the C-terminus or in hydrophilic regions,is described by Ausubel et al. (supra), and others.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems 431A peptide synthesizer usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio iodinated, goat anti-rabbitIgG.

XII Purification of Naturally Occurring HMTP Using Specific Antibodies

Naturally occurring or recombinant HMTP is substantially purified byimmunoaffinity chromatography using antibodies specific for HMTP. Animmunoaffinity column is constructed by covalently coupling HMTPantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing HMTP is passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof HMTP (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/HMTP binding (eg, a buffer of pH 2-3 or a high concentration ofa chaotrope, such as urea or thiocyanate ion), and HMTP is collected.

XIII Identification of Molecules Which Interact with HMTP

HMTP or biologically active fragments thereof are labeled with ¹²⁵ IBolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled HMTP, washed and any wells withlabeled HMTP complex are assayed. Data obtained using differentconcentrations of HMTP are used to calculate values for the number,affinity, and association of HMTP with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 3                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 310 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -    (vii) IMMEDIATE SOURCE:                                                            (A) LIBRARY: ISLTNOT01                                                        (B) CLONE: 2075107                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Met Phe Trp Arg Arg Leu Asn Glu Ala Ala Va - #l Thr Val Ser Arg Glu         #                15                                                           - Ala Thr Thr Leu Thr Ile Val Phe Ser Gln Le - #u Pro Leu Pro Ser Pro         #            30                                                               - Gln Glu Thr Gln Lys Phe Cys Glu Gln Val Hi - #s Ala Ala Ile Lys Ala         #        45                                                                   - Phe Ile Ala Val Tyr Tyr Leu Leu Pro Lys As - #p Gln Gly Ile Thr Leu         #    60                                                                       - Arg Lys Leu Val Arg Gly Ala Thr Leu Asp Il - #e Val Asp Gly Met Ala         #80                                                                           - Gln Leu Met Glu Val Leu Ser Val Thr Pro Th - #r Gln Ser Pro Glu Asn         #                95                                                           - Asn Asp Leu Ile Ser Tyr Asn Ser Val Trp Va - #l Ala Cys Gln Gln Met         #           110                                                               - Pro Gln Ile Pro Arg Asp Asn Lys Ala Ala Al - #a Leu Leu Met Leu Thr         #       125                                                                   - Lys Asn Val Asp Phe Val Lys Asp Ala His Gl - #u Glu Met Glu Gln Ala         #   140                                                                       - Val Glu Glu Cys Asp Pro Tyr Ser Gly Leu Le - #u Asn Asp Thr Glu Glu         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Asn Asn Ser Asp Asn His Asn His Glu Asp As - #p Val Leu Gly Phe Pro         #               175                                                           - Ser Asn Gln Asp Leu Tyr Trp Ser Glu Asp As - #p Gln Glu Leu Ile Ile         #           190                                                               - Pro Cys Leu Ala Leu Val Arg Ala Ser Lys Al - #a Cys Leu Lys Lys Ile         #       205                                                                   - Arg Met Leu Val Ala Glu Asn Gly Lys Lys As - #p Gln Val Ala Gln Leu         #   220                                                                       - Asp Asp Ile Val Asp Ile Ser Asp Glu Ile Se - #r Pro Ser Val Asp Asp         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Ala Leu Ser Ile Tyr Pro Pro Met Cys Hi - #s Leu Thr Val Arg Ile         #               255                                                           - Asn Ser Ala Lys Leu Val Ser Val Leu Lys Ly - #s Ala Leu Glu Ile Thr         #           270                                                               - Lys Ala Ser His Val Thr Pro Gln Pro Glu As - #p Ser Trp Ile Pro Leu         #       285                                                                   - Leu Ile Asn Ala Ile Asp His Cys Met Asn Ar - #g Ile Lys Glu Leu Thr         #   300                                                                       - Gln Ser Glu Leu Glu Leu                                                     305                 3 - #10                                                   - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1540 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -    (vii) IMMEDIATE SOURCE:                                                            (A) LIBRARY: ISLTNOT01                                                        (B) CLONE: 2075107                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - AACGAGCCCT TGACGCTGTG GCCCGGAAGT GGAGCGGCTG TCGCAGTGCG GC - #TCCGGCAG         60                                                                          - TGGCAGCGGA GGCCTGTGTT TGCGGCCTTC GGCAAGCGAC TGAGATGGCG AG - #CGCAACTG        120                                                                          - CACCTGCAGC CGCAGTCCCC ACCCTGGCTT CGCCTTTGGA GCAGCTCCGG CA - #CTTGGCGG        180                                                                          - AGGAGCTGCG GTTGCTCCTG CCTCGAGTGC GGGTCGGCGA ACCCAGGAGA CC - #ACCGAGGA        240                                                                          - GTTTAATCGA GAGATGTTCT GGAGAAGACT CAATGAGGCA GCTGTGACTG TG - #TCAAGGGA        300                                                                          - AGCCACGACT CTGACCATAG TCTTCTCTCA GCTTCCACTG CCGTCTCCAC AG - #GAAACCCA        360                                                                          - GAAGTTCTGT GAACAAGTCC ATGCTGCCAT CAAGGCATTT ATTGCAGTGT AC - #TATTTGCT        420                                                                          - TCCAAAGGAT CAGGGGATCA CCCTGAGAAA GCTGGTACGG GGCGCCACCC TG - #GACATCGT        480                                                                          - GGATGGCATG GCTCAGCTCA TGGAAGTACT TTCCGTCACT CCAACTCAGA GC - #CCTGAGAA        540                                                                          - CAATGACCTT ATTTCCTACA ACAGTGTCTG GGTTGCGTGC CAGCAGATGC CT - #CAGATACC        600                                                                          - AAGAGATAAC AAAGCTGCAG CTCTTTTGAT GCTGACCAAG AATGTGGATT TT - #GTGAAGGA        660                                                                          - TGCACATGAA GAAATGGAGC AGGCTGTGGA AGAATGTGAC CCTTACTCTG GC - #CTCTTGAA        720                                                                          - TGATACTGAG GAGAACAACT CTGACAACCA CAATCATGAG GATGATGTGT TG - #GGGTTTCC        780                                                                          - CAGCAATCAG GACTTGTATT GGTCAGAGGA CGATCAAGAG CTCATAATCC CA - #TGCCTTGC        840                                                                          - GCTGGTGAGA GCATCCAAAG CCTGCCTGAA GAAAATTCGG ATGTTAGTGG CA - #GAGAATGG        900                                                                          - GAAGAAGGAT CAGGTGGCAC AGCTGGATGA CATTGTGGAT ATTTCTGATG AA - #ATCAGCCC        960                                                                          - TAGTGTGGAT GATTTGGCTC TGAGCATATA TCCACCTATG TGTCACCTGA CC - #GTGCGAAT       1020                                                                          - CAATTCTGCG AAACTTGTAT CTGTTTTAAA GAAGGCACTT GAAATTACAA AA - #GCAAGTCA       1080                                                                          - TGTGACCCCT CAGCCAGAAG ATAGTTGGAT CCCTTTACTT ATTAATGCCA TT - #GATCATTG       1140                                                                          - CATGAATAGA ATCAAGGAGC TCACTCAGAG TGAACTTGAA TTATGACTTT TC - #AGGCTCAT       1200                                                                          - TTGTACTCTC TTCCCCTCTC ATCGTCATGG TCAGGCTCTG ATACCTGCTT TT - #AAAATGGA       1260                                                                          - GCTAGAATGC TTGCTGGATT GAAAGGGAGT GCCTATCTAT ATTTAGCAAG AG - #ACACTATT       1320                                                                          - ACCAAAGATT GTTGGTTAGG CCAGATTGAC ACCTATTTAT AAACCATATG CG - #TATATTTT       1380                                                                          - TCTGTGCTAT ATATGAAAAA TAATTGCATG ATTTCTCATT CCTGAGTCAT TT - #CTCAGAGA       1440                                                                          - TTCCTAGGAA AGCTGCCTTA TTCTCTTTTT GCAGTAAAGT ATGTTGTTTT CA - #TTGTAAAG       1500                                                                          #  1540            TAAA ATGCTAACTT GCCAGTGATT                                 - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 310 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -    (vii) IMMEDIATE SOURCE:                                                            (A) LIBRARY: GenBank                                                          (B) CLONE: 1255172                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Met Phe Trp Arg Arg Leu Asn Glu Ala Ala Va - #l Lys Val Asn Gly Glu         #                15                                                           - Ala Thr Val Leu Thr Thr His Phe Ser Lys Le - #u Pro Trp Pro Ser Pro         #            30                                                               - Gln Glu Thr Gln Arg Ile Cys Glu Gln Val Ar - #g Ile Ala Ile Glu Glu         #        45                                                                   - Ile Ile Ile Val Tyr Tyr Ser Leu Pro Lys As - #p Gln Gly Ile Thr Leu         #    60                                                                       - Arg Lys Leu Val Arg Asn Ala Ala Leu Asp Il - #e Val Asp Gly Met Ala         #80                                                                           - Gln Leu Leu Glu Val Leu Leu Thr Ala Pro Se - #r Gln Ser Thr Glu Asn         #                95                                                           - Gly Asp Leu Ile Ser Cys Asn Ser Val Ser Va - #l Ala Cys Gln Gln Val         #           110                                                               - Pro Glu Ile Pro Lys Asp Asn Lys Ala Ala Al - #a Leu Leu Met Leu Thr         #       125                                                                   - Lys Ser Val Asp Phe Val Lys Asp Ala His Gl - #u Glu Met Glu Gln Ala         #   140                                                                       - Val Glu Glu Cys Asp Pro Tyr Ser Gly Leu Le - #u Asn Asp Ser Glu Asp         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Asn Ser Asp Ser His Ser Asp Glu Asp Gly Va - #l Leu Gly Leu Pro Ser         #               175                                                           - Asn Arg Asp Ser Tyr Trp Ser Glu Glu Asp Gl - #n Glu Leu Ile Ile Pro         #           190                                                               - Cys Leu Ala Leu Val Arg Ala Ser Arg Ala Se - #r Leu Lys Lys Ile Arg         #       205                                                                   - Ile Leu Val Ala Glu Asn Gly Lys Lys Asp Gl - #u Val Ala Gln Leu Asp         #   220                                                                       - Asp Ile Val Gly His Phe Leu Met Arg Ser Al - #a Pro Val Trp Ile Asp         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Leu Val Leu Ser Ile Tyr Pro Pro Val Cys Hi - #s Leu Thr Val Arg Ile         #               255                                                           - Ser Ser Ala Lys Leu Val Ser Val Leu Ile Ly - #s Ala Leu Glu Ile Thr         #           270                                                               - Lys Ala Ser His Val Ser Pro His Pro Gly As - #p Ser Trp Ile Pro Leu         #       285                                                                   - Leu Ile Asn Ala Val Asp His Cys Met Asn Ar - #g Ile Lys Ala Leu Thr         #   300                                                                       - Gln Arg Ala Ala Glu Leu                                                     305                 3 - #10                                                   __________________________________________________________________________

What is claimed is:
 1. An isolated and purified polynucleotide encodinga polypeptide comprising the amino acid sequence of SEQ ID NO:1.
 2. Acomposition comprising the polynucleotide of claim
 1. 3. An isolated andpurified polynucleotide which hybridizes under stringent wash conditionsof 0.1× saline sodium citrate and 0.5% sodium dodecyl sulfate to thepolynucleotide of claim
 1. 4. An isolated and purified polynucleotidewhich is complementary to the polynucleotide of claim
 1. 5. Anexpression vector containing the polynucleotide of claim
 1. 6. A hostcell containing the expression vector of claim
 5. 7. A method forproducing a polypeptide comprising the amino acid sequence of SEQ IDNO:1, the method comprising the steps of:a) culturing the host cell ofclaim 6 under conditions for the expression of the polypeptide; and b)recovering the polypeptide from the host cell culture.
 8. A method fordetecting a polynucleotide which encodes a human maternally transcribedprotein in a biological sample comprising the steps of:a) hybridizingthe polynucleotide of claim 4 to nucleic acid material of a biologicalsample, thereby forming a hybridization complex; and b) detecting saidhybridization complex, wherein the presence of said complex correlateswith the presence of a polynucleotide encoding human maternallytranscribed protein in said biological sample.
 9. The method of claim 8wherein the nucleic acid material is amplified by polymerase chainreaction prior to hybridization.
 10. An isolated and purifiedpolynucleotide comprising SEQ ID NO:2.
 11. A composition comprising thepolynucleotide of claim
 10. 12. An isolated and purified polynucleotidewhich is complementary to the polynucleotide of claim 10.